Method for controlling hemophilia in mammals

ABSTRACT

A method for controlling HemophiliaA (Antihemophilic Factor (F. VIII:C) deficiency) in mammals is described. Factor VIII:C deficiency in the mammal is bypassed by infusion of a synergistic mixture of a phospholipid and Factor Xa so that the cascade process of blood clotting may continue. The proportions of phospholipid and Factor Xa in the mixture are critical as too little Xa has no effect while too much is toxic (thrombogenic).

FIELD OF INVENTION

This invention relates to the control of hemophilic bleeding in mammals.More particularly this invention relates to the therapeutic treatment ofFactor VIII:C deficient patients by means of a Factor VIII:C bypasstechnique using a synergistic mixture of phospholipids with Factor Xa.

BACKGROUND OF INVENTION

Classic Hemophilia A is a sex-linked recessive inherited disorder of theblood where the activity of a specific coagulation factor (protein),required for the cascade or chain process for blood coagulation, iseither reduced or absent. Hemophilia afflicts about 1 in 10,000 of themale population. This produces a severe bleeding disorder andconstitutes the most frequently clinically encountered congentialcoagulation disorder. Since about 1965 the prognosis of affectedindividuals has considerably improved due to the availability ofspecific clotting factor replacement products derived from the blood ofnormal donors which can be transfused. These products contain the mostusually absent factor, Factor VIII in a concentrated form.Unfortunately, however, approximately 10% of all treated hemophiliacsdevelop antibodies to the transfused Factor VIII:C and becomeuntreatable by this means. It is an aim of the present invention toprovide a method for the treatment and management of such antibodysensitized hemophiliacs.

DISCUSSION OF PRIOR ART

Heretofore hemophiliacs with antibodies to F. VIII:C have been managedby various therapies none of which are satisfactory. The use ofimmunosuppressive therapy is morbidity. The use of Factor VIII:C derivedfrom other species, i.e. porcine or bovine, has been shown to be aneffective replacement but may be associated with major side effects dueto the development of heterologous antibodies. Recently considerableinterest has been shown in using prothrombin complex concentrates (PCC).As explained in more detail hereinafter, blood coagulation proceeds by aseries or cascade of activation steps where circulating inactiveclotting factors (zymogens) are converted to proteolytic enzymes. Thefinal product of the cascade is thrombin (IIa) which converts the solprotein, fibrinogen, to its gel form, fibrin. Recent work hasdemonstrated that Factor VIII:C is not a proteolytic enzyme but a potentco-factor of the activation step whereby Factor IXa activates Factor Xto Xa. In classic Hemophilia A, this co-factor activity is reduced ormissing so that insignificant activation of Factor X takes place despiteall other clotting factors being present at normal levels. As notedabove, transfusion of Factor VIII:C concentrates can correct thisabnormality and similar concentrates have been developed for thecongenital deficiency of Factor IX. These concentrates differ from thoseof Factor VIII:C in containing significant quantities of other clottingfactors namely X, VII and II (prothrombin). Moreover, it is the rulethat all concentrates contain trace contaminents of the activatedproducts of these clotting factors, namely IXa, Xa, VIIa and IIa(thrombin). It will be noted that, with the exception of Factor IX, theremaining three clotting factors are placed in the cascade below thecritically important Factor VIII:C-dependent step. It has beenpostulated, that these concentrates, by providing pre-formed activatedproducts, may achieve Factor VIII:C bypassing activity (FEBA) inhemophiliacs where Factor VIII:C replacement is precluded by thedevelopment of antibodies to this clotting factor. Initial anecdotalclinical reports were promising but by no means unanimous. This lack ofunanimity related to the uncertainty as to which, if any, of thecomponent clotting factors were the most critical. The products used areof two types. The first are known as "unactivated" PCC and are productsthat have been developed specifically to replace deficiencies of theclotting factors that they contain. In such patients, it is consideredundesirable to infuse preactivated clotting factors because of concernfor thromboembolic side effects. Therefore, attempts are made, in thefractionation process, to minimize the activated clotting factor contentalthough all products contain some. As it was the activated clottingfactor content that was considered to be the putative agent(s) in thetreatment of hemophiliacs with inhibitors, some manufacturers havedeliberately activated the PCC preparations for this purpose. These areknown as "activated" PCC. Recent clinical trials have confirmed thebenefit of the use of non-activated PCC as compared with placebo but theresponse was less than optimal in comparison to that which would beexpected from conventional Factor VIII:C replacement in hemophiliacswithout inhibitors. A similar study compared treatment with anunactivated PCC with an activated PCC prepared by the same manufacturer.There appeared to be a marginal benefit in favour of the activatedpreparation. Despite this, the response remained suboptimal and theabsence of any clear indication as to the specific constituent of thepreparation responsible for the effect seen, it is impossible to ensureinter-batch reproducibility of individual production lots of apparentlythe same product. As a result, there is still not universal agreement asto the validity of this approach.

In "Blood", v. 59, p. 401-407, Feb. 1982, Giles et al demonstrated thatthe in vivo thrombogenicity of prothrombin complex concentrates washighly correlated with their individual content of coagulant activephospholipid. However, this component alone was nonthrombogenic butrequired the presence of Factor Xa. At high dose, the latter wasthrombogenic alone but its potency was drastically increased in thepresence of small amounts of coagulant-active phospholipid. It wassuggested that the combination of these two components accounted for thethrombogenicity associated with the use of prothrombin complexconcentrates and evidence was presented that this thrombogenic effectcould be mimicked by a combination of highly purified Factor Xa andphosphatidylcholinephosphatidylserine (PCPS) lipid vesicles. Thisconfirmed the findings of Barton et al in the Journal of Lipid Researchv. 11, p. 87, 1970, who used less well-defined protein/lipid components.

SUMMARY OF INVENTION

As can be seen from FIG. 1, the presence of Factor Xa alone bypasses therequirement for Factor VIII:C. Although this is the case in vitro, invivo the presence of a number of inhibitory processes complicate its usein achieving Factor VIII:C by passing activity. Combining this factorwith coagulant-active phospholipid in the form of PCPS vesicles exertsan apparent synergistic activity in vivo presumably by mimicking thenormal interactions between Factor Xa and platelet phospholipid. It hasbeen demonstrated that the dose of each component administered iscritical in that a minimum dose of Factor Xa/kg body weight is requiredand that the dose of phospholipid must be limited to avoid unacceptabletoxicity (thrombogenicity).

Thus, by one aspect of this invention there is provided a method forcontrolling hemophilia in mammals comprising administering intravenouslyto said mammal a synergistic mixture of phospholipid vesicles andmammalian blood Factor Xa in relative proportions and in an amount justsufficient to arrest bleeding.

By another aspect of this invention there is provided a pharmaceuticalcomposition for the treatment of hemophilia in mammals comprising asynergistic mixture of phospholipid vesicles and mammal blood Factor Xain relative proportions just sufficient to arrest bleeding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the stages in the cascadeprocess of blood clotting.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIG. 1, blood coagulation proceeds by a series of activationsteps where circulating inactive clotting factors are converted toproteolytic enzymes. The final product of this cascade is thrombin (IIa)which converts the sol protein, fibrinogen, to its gel form fibrin whichis the basic constituent of a blood clot. Factor VIII:C is not aproteolytic enzyme but a potent co-factor of the activation step inwhich Factor IXa activates Factor X to Xa. Indeed Factor VIII:C is arate limiting factor, in the absence or reduction of which activation ofFactor X to Factor Xa is prevented or minimized even in the presence ofnormal levels of all other clotting factors. It is known that thecomplex of Factor Xa, Factor V and calcium responsible for theconversion of prothrombin (Factor II) to thrombin (Factor IIa) isassembled on a phospholipid surface provided by the platelet. It isproposed that the synergistic effect of highly-purified factor Xa incombination with PCPS vesicles provides a close approximation of thephysiological event. The remaining components of the complex, i.e.Factor V and ionized calcium, being unaffected by the availability ofFactor VIII:C, are available in the recipient's blood. Studies havedemonstrated true synergism between the two components, i.e. Factor Xaand PCPS vesicles, in vivo. Decreasing the dose of Factor Xa can beaccommodated by increasing the dose of PCPS and vice versa in achievingthe same end-point, i.e. thrombin generation. It is now known, however,that thrombin has multiple roles in vivo and some of these are mutuallyantagonistic. As shown in FIG. 1, thrombin is the proteolytic enzymerequired for the conversion of fibrinogen to fibrin. It is also knownthat thrombin is required to activate a Vitamin K-dependent protein,Protein C, which is a potent anticoagulant. This anticoagulant effect isachieved by the inactivation of the critically important cofactors,Factors VIII:C and V. It also appears to exert significant control overthe fibrinolytic mechanism, i.e. the mechanism responsible for clearingfibrin formed by the conversion of fibrinogen by thrombin. It has beendemonstrated that activated Protein C requires a phospholipid surface inorder to exert its anticoagulant effect. Consequently, the Factor VIII:Cbypassing effect, in achieving hemostasis, requires a critical doseratio of Factor Xa to PCPS. This is calculated on a dose/kg body weightbasis. The dose of Factor Xa is critical. Above a given level,unacceptable toxicity (thrombogenicity) occurs whereas below a certainlevel, a hemorrhagic tendency is produced in normal animals, i.e. VIII:Creplete animals, presumably due to the relative excess of phospholipidfavouring the anticogulant effect of activated Protein C.

Factor Xa may be obtained by fractionating plasma from normal donors toobtain the precursor zymogen Factor X which can then be activated byknown procedures (Bajaj et al. J. Biol. Chem. 248:7729, 1973; Downing etal. J. Biol. Chem. 250:8897, 1975). Factor Xa may be stored indefinitelyin 50% glycerol at -20° C. The amount of Factor Xa in the dosage form isextremely small and sufficient quantities to treat large numbers ofhemophiliacs can be derived from a small number of blood donors, incomparison to the many thousands required for the provision of moreconventional therapy. This has distinct advantages, apart from theobvious one of economy. The accidental transmission of infection is amajor hazard of multiple transfusion practice in patients such ashemophiliacs. Hepatitis and acquired immunodeficiency syndrome are majorproblems. By restricting the number of donors required, carefulscreening for these problems may be effected thus drastically reducingif not eliminating the risk. Furthermore, the purified Factor Xa, unlikemany of the blood products presently used can be sterilized withrelative ease, and in comparison to Factor VIII:C is relatively stablethus making it more suitable for use in areas where sophisticatedhospital facilities are not available.

Phosphatidylcholine and phosphatidylserine are available commercially assemi-purified reagents. They are prepared from egg yolks and bovinebrain respectively. The PCPS lipid vesicles may be prepared by aconventional and standardized protocol (Nesheim et al, J. Biol. Chem.254: 10952, 1979 and Barenholz et al Biochem. J. 16:2806, 1976) whichproduces single compartment vesicles of uniform dimension (325-350 Å)which may be stored at 4° C. for 2 to 3 weeks. The molar ratio ofphosphatidylserine to phosphatidylcholine is about 1:3, based on therelative amounts of these lipids used in the preparation of thevesicles.

The Factor Xa-PCPS mixture is freshly prepared by mixing Factor Xa andPCPS in the desired ratio immediately prior to use.

In order to demonstrate the efficacy of the treatment tests were carriedout on both normal and specially bred hemophilic dogs, maintained onwater ad libitum and regular dry dog Chow (Ralston-Purina, St. Louis,Mo.), as described in detail in Examples 1-7 hereinafter. The animalswere anesthetized with a rapid acting intravenous barbiturate 5%-18mg/kg body weight. A continuous infusion was established via a 21 gaugebutterfly needle in the cephalic vein using isotonic saline forinjection to keep the vein open. All medications were administered viathis route. All hair was clipped from around the animal's claws andsilicone grease was applied to prevent blood from tracking back beneaththe claw. A spring loaded sliding blade guillotine was used to sever theapex of the nail cuticle which was visualized or located in relation tothe dorsal nail groove. Blood was allowed to fall freely by positioningthe paw over the edge of the operating table. In normal dogs bleedingstops abruptly (mean 6.0±3.7 (S.D.) mins) whereas in hemophilic animalsbleeding may stop transiently but always restarts and continues untilarrested by the application of silver nitrate (Blood, Vol. 60, No. 3,P727-730 Sept. 1982). In all cases the dosage of Factor Xa/PCPS wasadministered on a dose/kg body weight basis. The dose of PCPS isunitized in arbitrary units. 1 Arbitrary unit PCPS equals 1×10⁻⁸ molesof phospholipid as assayed by an inorganic phosphorus assay. Factor Xais unitized according to an internationally accepted classification inwhich 1 unit of Factor X is the amount present in 1 ml of normal plasmaand 1 unit of Factor Xa is the amount of activity present when 1 unit ofFactor X is fully activated. The assay is standardized by measuringactivity in the test preparation against the activity in a normal poolplasma standard as described by Suomela H et al (Thrombosis Research10:267, 1977) as modified by Giles A. R. et al (Thrombosis Research 17;353, 1980).

EXAMPLE 1

A normal dog was tested as described above, by cutting the cuticle ofthe right hind nail 4. Bleeding stopped spontaneously at 5 minutes butrebleeding occurred at 9 minutes for a further 3 minutes. 15 Minutesafter the start of the first cuticle bleeding time, the animal wasinfused with PCPS/Xa at a dose of 40 units and 0.05 units/kg body weightrespectively. 2 Minutes after this infusion, the right hind nail 3 wassevered. Bleeding continued for 12 minutes and the cuticle requiredcautery with silver nitrate application. 60 Minutes after the infusionof PCPS/Xa, the right hind nail 2 was severed but bleeding ceasedspontaneously after 7 minutes. It will be noted that the cuticlebleeding time was initially normal but became abnormal immediately afterthe infusion of PCPS/Xa at this dosage suggesting that the relativeexcess of PCPS had favoured the anticoagulant effect of activatedProtein C, thus compromising the generation of fibrin normally requiredto cause bleeding to stop. This effect had dissipated 60 minutes afterthe infusion of PCPS/Xa.

EXAMPLE 2

This procedure of Example 1, showing the effect of PCPS/Xa at a dosageof 40 units and 0.05 units/kg body weight respectively on the cuticlebleeding time of a normal dog was repeated. The cuticle of the leftfront nail 1 was severed and bleeding arrested spontaneously after 3minutes. 13 Minutes after the start of the first cuticle bleeding time,a bolus infusion of PCPS/Xa was given at a dosage of 40 units/0.05units/kg body weight. 2 Minutes after the infusion, left front nail 3was severed and bleeding continued for 12 minutes until arrested bysilver nitrate cautery. 60 Minutes after the infusion of PCPS/Xa, leftfront nail 2 was severed and bleeding arrested spontaneously after 3minutes. The results obtained are virtually identical to those given inExample 1 and the same conclusion is drawn.

EXAMPLE 3

The same cuticle bleeding time procedure as in Examples 1 and 2 was usedin a hemophilic dog (Factor VIII:C level <4%). The right front nail 4was severed and bleeding continued for 14 minutes until PCPS/Xa at adosage of 8.0 units and 0.2 units/kg body weight respectively wasinfused as a bolus. Bleeding stopped abruptly but 2 small rebleeds (1drop in each case) occurred at 18 and 23 minutes post the start ofcuticle bleeding time number 1. 1 Minute prior to the infusion ofPCPS/Xa, the right front nail 3 was severed but bleeding arrested 30seconds after the administration of PCPS/Xa. It should be noted that theobservation period was not continued beyond 30 minutes post the start ofthe cuticle bleeding time number 1. These results demonstrate that thecombination of PCPS/Xa, at the dosage used, bypasses Factor VIII incausing the arrest of bleeding in a Factor VIII:C deficient animal. Theinjured cuticles of such animals would normally bleed until cauterizedwith silver nitrate. Immediately following the infusion, the animalexhibited apnea and cardiac rhythm irregularities but these resolvedspontaneously within 5 minutes after the infusion.

EXAMPLE 4

The procedure of Example 3 was repeated using a different hemophilicanimal (Factor VIII:C <1%). The right hind nail 1 was severed andbleeding continued until a bolus infusion of PCPS/Xa at a dosage of 8.0units and 0.2 units/kg body weight respectively was administered at 16minutes post the start of the cuticle bleeding time number 1. 1 Minuteprior to the administration of PCPS/Xa, the right hind nail 2 wassevered but bleeding arrested within 30 seconds of administration ofPCPS/Xa. Rebleeding occurred 12 minutes later and continued for 17minutes until arrested by silver nitrate cautery. 18 Minutes after theadministration of PCPS/Xa, the right hind nail 3 was severed andbleeding continued for 12 minutes until arrested by silver nitratecautery. Immediately following the infusion of PCPS/Xa the animal had atransient cardiopulmonary arrest but regained his vital signs within 2minutes without resuscitation other than the application of 100% oxygenvia a face mask. These results confirm the Factor VIII bypassingactivity of a combination of PCPS/Xa at this dosage. The cardiopulmonaryside-effects suggest borderline toxicity at this dosage. The rebleedingof right hind nail 2 and the abnormal cuticle bleeding time of righthind nail 3 suggests that the Factor VIII:C bypassing effect istransitory.

EXAMPLE 5

A hemophilic dog, as in Examples 3 and 4 was tested as described above.The right hind nail 2 was severed and bleeding from the cuticle stoppedwith silver nitrate after 12 minutes. The left hind nail 1 was severedand 2 minutes thereafter PCPS/Xa at a dosage of 4.0 units and 0.1units/kg body weight respectively was infused. Bleeding continued for afurther 10 minutes until arrested by silver nitrate cautery. The animaldid not exhibit toxicity but bleeding was not arrested by this dosage ofPCPS/Xa.

EXAMPLE 6

The procedure followed in Example 5 was repeated in a differenthemophilic dog and with the dose of Factor Xa increased to 0.2 units/kgbody weight in combination with PCPS at a dose of 4.0 units/body weight.The right hind nail 1 was severed and bleeding continued for 15 minutesuntil the PCPS/Xa combination was administered as a bolus infusion.Bleeding stopped abruptly but reoccurred at 27 minutes and continuedthereafter until 40 minutes when it was arrested by silver nitratecautery. 1 Minute prior to the infusion of PCPS, the right hind nail 2was severed but bleeding ceased within 20 seconds of the infusion ofPCPS/Xa combination. Bleeding recommenced 12 minutes later and continuedfor 13 minutes until arrested with silver nitrate cautery. Nocardiopulmonary toxicity was observed. As compared with the result ofExamples 3 and 4, a 50% reduction of the dosage of PCPS did notcompromise the Factor VIII:C bypassing activity observed in thehemophilic animals. In comparison with Example 5, doubling the dose ofFactor Xa in combination with 4 units of PCPS achieved the Factor VIII:Cbypass not achieved by the lower dosage of Factor Xa in combination withthe same dosage of PCPS.

EXAMPLE 7

The procedure of Example 6 was repeated on another hemophilic dog withthe dosage of Factor Xa being maintained but the dosage of PCPS beingfurther reduced to 0.1 unit/kg body weight. The left front nail 5 wassevered and bleeding continued for 16 minutes but was arrested abruptlyby the infusion of PCPS/Xa. No bleeding occurred during the period ofobservation. 1 Minute prior to the infusion of PCPS/Xa, the left frontnail 4 was severed and bleeding was arrested within 1 minute of theinfusion of PCPS/Xa but reoccurred 4 minutes later and continued for 22minutes until arrested by silver nitrate cautery. 15 Minutes after theinfusion of PCPS/Xa, the left front nail 3 was severed and bleedingcontinued for 12 minutes until arrested by silver nitrate cautery. Theseresults show that a further significant reduction in PCPS/Xa dosage isstill associated with Factor VIII bypassing activity but that the effectis less well maintained.

On the basis of these studies, all of which have been carried out on adog model using Factor Xa derived from bovine blood, it is believed thatthe minimum dose of Factor Xa required is 0.2 units/kg body weight inconjunction with not less than 1 unit/kg body weight of PCPS vesicles.Tests have confirmed similar results using Factor Xa derived from canineblood and human blood. Infusion of either of these components alone havebeen shown to have no effect in correcting the cuticle bleeding time ofFactor VIII:C deficient animals nor are they thrombogenic. However, itis stressed that Factor Xa in combination with PCPS is an extremelypotent reagent and as little as 0.5 units/kg body weight may besufficiently toxic, i.e. thrombogenic, to cause death. As thecombination of Factor Xa and PCPS is synergistic, lower doses of FactorXa may become toxic (i.e. thrombogenic) when combined with higher dosesof PCPS. The examples given suggest that threshold toxicity of Factor Xaat a dosage of 0.02 units/kg body weight is achieved when combined withPCPS at a dosage of 8 units/kg body weight. This ratio of PCPS to Xa(40:1) is considered to be the practical maximum whereas the ratio ofPCPS 1 unit to Xa 0.2 units (5:1) is considered to be the practicalminimum.

It is also emphasized that these studies have been carried out on a dogmodel and although this model is believed to simulate the human diseaseof classical Hemophilia A (Factor VIII:C deficiency) very closely, thespecific ratios between, and the actual dosage of, PCPS and factor Xa inthe synergistic mixture thereof may vary somewhat from those determinedin the dog model.

We claim:
 1. A method for controlling hemophilia in mammals comprisingadministering intravenously to a hemophilic said mammal a synergisticmixture of phospholipid vesicles and mammalian blood factor Xa inrelative proportion and in an amount just sufficient to arrest bleeding.2. A method as claimed in claim 1 wherein said hemophilic mammal is adog.
 3. A method as claimed in claim 2 wherein said phospholipidvesicles are phosphatidylcholine and phosphatidylserine (PCPS).
 4. Amethod as claimed in claim 3 wherein said PCPS is present in saidmixture in an amount between about 1 and 8 units per kg of body weight.5. A method as claimed in claim 4 wherein Factor Xa is present in saidmixture in an amount between about 0.2 and less than 0.5 unit per kg ofbody weight.
 6. A method as claimed in claim 3 wherein the ratio of PCPSto Factor Xa is in the range 40:1 to 5:1.